VEGF对体外培养绒山羊次级毛囊外根鞘细胞的影响
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摘要
本研究旨在探讨陕北白绒山羊毛囊外根鞘细胞的培养和VEGF对次级毛囊外根鞘细胞增殖、分化及细胞中增殖细胞核抗原(PCNA)、角蛋白10(K10)和成纤维细胞生长因子5(FGF5)mRNA表达量的影响。采集绒山羊体侧部皮肤,并利用消化等方法分离次级毛囊外根鞘细胞,置于37℃、5%CO_2、饱和湿度条件下培养;选用第5代次级毛囊外根鞘细胞,添加不同质量浓度血管内皮生长因子(VEGF),分别处理24、48h,噻唑兰比色法(MTT)检测细胞活力,EdU核酸标记技术检测细胞增殖;实时荧光定量PCR技术检测PCNA、K 10、FGF5mRNA的表达情况。结果显示:1)成功培养出次级毛囊外根鞘细胞,CK17和CK15细胞免疫组化呈阳性。2)VEGF可以促进绒山羊次级毛囊外根鞘细胞的增殖。相同浓度VEGF处理48h组的细胞活力极显著高于处理24h组(P<0.01);在48h处理组中,100ng·mL~(-1)组的细胞活力极显著高于对照组、1ng·mL~(-1)组、10ng·mL~(-1)组(P<0.01),并显著高于50ng·mL~(-1)组(P<0.05),50ng·mL~(-1)组的细胞活力显著高于对照组(P<0.05);100ng·mL~(-1)组和50ng·mL~(-1)组增殖细胞比例均极显著高于对照组、1ng·mL~(-1)组、10ng·mL~(-1)组(P<0.01),100ng·mL~(-1)组显著高于50ng·mL~(-1)组(P<0.05)。3)PCNA、K10、FGF5mRNA在次级毛囊外根鞘细胞中均有表达。在VEGF处理48h时,PCNA mRNA的表达量100ng·mL~(-1)组最高,极显著高于对照组(P<0.01),显著高于10ng·mL~(-1)组(P<0.05);K10mRNA的表达量对照组最高,极显著高于10ng·mL~(-1)组与100ng·mL~(-1)组(P<0.01);FGF5mRNA的表达量对照组最高,极显著高于100ng·mL~(-1)组(P<0.01)。综上表明,本研究成功分离培养了陕北白绒山羊次级毛囊外根鞘细胞;VEGF能够促进次级毛囊外根鞘细胞的增殖,抑制细胞的分化;VEGF可能通过抑制次级毛囊外根鞘细胞中FGF5基因的表达而促进毛囊生长。
This study was conducted to investigate isolation and cultivation of the hair follicle outer root sheath cells(HFORSCs)in vitro,and to estimate the effects of the vascular endothelial growth factor(VEGF)on the proliferation,differentiation and mRNA expression of proliferating cell nuclear antigen(PCNA),keratin 10(K10)and fibroblast growth factor 5(FGF5)genes in the sHFORSCs of Shaanbei White cashmere goat.Skin tissues of goats were collected,and sHFOR-SCs were obtained through the methods of digestion and then cultured under the condition of37 ℃,5% CO_2 and 100% relative humidity;The 5 th generation of cultured sHFORSCs were selected and treated with different concentration VEGF for 24 and 48 h,respectively.The cell viability was detected by Methyl thiazolyl tetrazolium(MTT),and the cell proliferation was detected by EdU DNA markers;The mRNA expression levels of PCNA,K 10 and FGF 5 in HFORSCs were detected by the real-time fluorescence quantitative PCR.Results showed that:1)sHFORSCs were successfully isolated and cultured in vitro,and cultured cells were positive in the immunohistochemistry test of CK17 and CK15,which were main biomarkers of outer root sheath cells.2)VEGF could promote the proliferation of sHFORSCs.Treated with same concentration of VEGF,the cell viability of 48 hgroup was significantly higher than that of 24 hgroup(P<0.01).Treated with different concentrations of VEGF for 48 h,the cell viability of 100 ng·mL~(-1) group was significantly higher than that of control group,1 ng·mL~(-1) group and 10 ng·mL~(-1) group(P<0.01),and also significantly higher than that of 50 ng·mL~(-1) group(P<0.05).The cell viability of 50 ng·mL~(-1) group was significantly higher than that of control group(P<0.05);The proportion of the cell proliferation of 100 and 50 ng·mL~(-1) groups were all significantly higher than that of control group,1 ng·mL~(-1) group and 10 ng·mL~(-1) group(P<0.01).The proportion of the cell proliferation of 100 ng·mL~(-1) group was significantly higher than that of 50 ng·mL~(-1) group(P<0.05).3)The expressions of mRNA of PCNA,K10 and FGF5 were all detected in HFORSCs.Treated with VEGF for 48 h,the mRNA expression level of PCNAin 100 ng·mL~(-1) group was the highest,and was significantly higher than that in control group(P<0.01)and 10 ng·mL~(-1) group(P<0.05);The mRNA expression of K10 in control group was the highest,and was significantly higher compared with that in 10 and 100 ng·mL~(-1) groups(P<0.01);The mRNA expression of FGF5 in control group was the highest,and was significantly higher than that in100 ng·mL~(-1) group(P<0.01).In this study,sHFORSCs were successfully isolated from hair follicle and were successfully cultured in vitro;The proliferation of HFORSCs could be promoted and the cell differentiation could be inhibited by VEGF.It inferred that VEGF might play the role of growth promotion of the hair follicle by inhibiting the mRNA expression of FGF5 in sHFORSCs.
This study was conducted to investigate isolation and cultivation of the hair follicle outer root sheath cells(HFORSCs)in vitro,and to estimate the effects of the vascular endothelial growth factor(VEGF)on the proliferation,differentiation and mRNA expression of proliferating cell nuclear antigen(PCNA),keratin 10(K10)and fibroblast growth factor 5(FGF5)genes in the sHFORSCs of Shaanbei White cashmere goat.Skin tissues of goats were collected,and sHFOR-SCs were obtained through the methods of digestion and then cultured under the condition of37 ℃,5% CO_2 and 100% relative humidity;The 5 th generation of cultured sHFORSCs were selected and treated with different concentration VEGF for 24 and 48 h,respectively.The cell viability was detected by Methyl thiazolyl tetrazolium(MTT),and the cell proliferation was detected by EdU DNA markers;The mRNA expression levels of PCNA,K 10 and FGF 5 in HFORSCs were detected by the real-time fluorescence quantitative PCR.Results showed that:1)sHFORSCs were successfully isolated and cultured in vitro,and cultured cells were positive in the immunohistochemistry test of CK17 and CK15,which were main biomarkers of outer root sheath cells.2)VEGF could promote the proliferation of sHFORSCs.Treated with same concentration of VEGF,the cell viability of 48 hgroup was significantly higher than that of 24 hgroup(P<0.01).Treated with different concentrations of VEGF for 48 h,the cell viability of 100 ng·mL~(-1) group was significantly higher than that of control group,1 ng·mL~(-1) group and 10 ng·mL~(-1) group(P<0.01),and also significantly higher than that of 50 ng·mL~(-1) group(P<0.05).The cell viability of 50 ng·mL~(-1) group was significantly higher than that of control group(P<0.05);The proportion of the cell proliferation of 100 and 50 ng·mL~(-1) groups were all significantly higher than that of control group,1 ng·mL~(-1) group and 10 ng·mL~(-1) group(P<0.01).The proportion of the cell proliferation of 100 ng·mL~(-1) group was significantly higher than that of 50 ng·mL~(-1) group(P<0.05).3)The expressions of mRNA of PCNA,K10 and FGF5 were all detected in HFORSCs.Treated with VEGF for 48 h,the mRNA expression level of PCNAin 100 ng·mL~(-1) group was the highest,and was significantly higher than that in control group(P<0.01)and 10 ng·mL~(-1) group(P<0.05);The mRNA expression of K10 in control group was the highest,and was significantly higher compared with that in 10 and 100 ng·mL~(-1) groups(P<0.01);The mRNA expression of FGF5 in control group was the highest,and was significantly higher than that in100 ng·mL~(-1) group(P<0.01).In this study,sHFORSCs were successfully isolated from hair follicle and were successfully cultured in vitro;The proliferation of HFORSCs could be promoted and the cell differentiation could be inhibited by VEGF.It inferred that VEGF might play the role of growth promotion of the hair follicle by inhibiting the mRNA expression of FGF5 in sHFORSCs.
引文
[1]SCHNEIDER M R,SCHMIDT-ULLRICH R,PAUS R.The hair follicle as a dynamic miniorgan[J].Curr Biol,2009,19(3):R132-R142.
[2]MULLER-ROVER S,FOITZIK K,PAUS R,et al.A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages[J].J Investig Dermatol,2001,117(1):3-15.
[3]KUMAMOTO T,SHALHEVET D,MATSUE H,et al.Hair follicles serve as local reservoirs of skin mast cell precursors[J].Blood,2003,102(5):1654-1660.
[4]KRAUSE K,FOITZIK K.Biology of the hair follicle:The basics[J].Semin Cutan Med Surg,2006,25(1):2-10.
[5]PENA J C,KELEKAR A,FUCHS E V,et al.Manipulation of outer root sheath cell survival perturbs the hair‐growth cycle[J].EMBO J,1999,18(13):3596-3603.
[6]PAUS R,COTSARELIS G.The biology of hair follicles[J].New Engl J Med,1999,341(7):491-497.
[7]LENOIR M C,BERNARD B A,PAUTRAT G,et al.Outer root sheath cells of human hair follicle are able to regenerate a fully differentiated epidermis in vitro[J].Dev Biol,1988,130(2):610-620.
[8]李京佳,林相国,许涛,等.VEGF家族及其在肿瘤生长中作用的研究[J].现代生物医学进展,2012,12(4):777-779,701.LI J J,LIN X G,XU T,et al.VEGF and the role of VEGF in tumor growth[J].Progress in Modern Biomedicine,2012,12(4):777-779,701.(in Chinese)
[9]蔡源源.血管内皮生长因子的调控及其作用研究进展[J].组织工程与重建外科杂志,2011,7(1):51-54.CAI Y Y.Advances in the regulation and role of VEGF[J].Journal of Tissue Engineering and Reconstructive Surgery,2011,7(1):51-54.(in Chinese)
[10]YANO K,BROWN L F,DETMAR M.Control of hair growth and follicle size by VEGF-mediated angiogenesis[J].J Clin Investig,2001,107(4):409-417.
[11]WU X J,ZHU J W,JING J,et al.VEGF165 modulates proliferation,adhesion,migration and differentiation of cultured human outer root sheath cells from central hair follicle epithelium through VEGFR-2activation in vitro[J].J Dermatol Sci,2014,73(2):152-160.
[12]袁超.绒山羊次级毛囊周期性变化相关microRNA的鉴定及miR-125b在毛乳头细胞中的功能研究[D].杨凌:西北农林科技大学,2014.YUAN C.Identification of microRNAs in secondary hair follicle cycling and functional analysis of miR-125bin dermal papilla cells of cashmere goats[D].Yangling:Northwest A&F University,2014.(in Chinese)
[13]周英昊,高晔,刘林丽,等.山羊y+L碱性氨基酸转运系统转录水平表达的研究[J].畜牧兽医学报,2015,46(8):1308-1316.ZHOU Y H,GAO Y,LIU L L,et al.Study on transcriptional level of y+L cationic amino acid transporter system in goat[J].Acta Veterinaria et Zootechnica Sinica,2015,46(8):1308-1316.(in Chinese)
[14]王丽,彭丽琴,张文彬,等.内蒙古白绒山羊皮肤毛囊发生发育规律的研究[J].畜牧兽医学报,1996,27(6):524-530.WANG L,PENG L Q,ZHANG W B,et al.Initiation and development of skin follicles in the inner mongolian cashmere goat[J].Acta Veterinaria et Zootechnica Sinica,1996,27(6):524-530.(in Chinese)
[15]刘志红,任立明,郭英,等.毛囊的周期性变化和分子调控及其在绒山羊上的研究进展[J].中国畜牧兽医,2009,36(4):103-107.LIU Z H,REN L M,GUO Y,et al.The cyclical variation and regulation of hair follicles and the study on cashmere goat[J].China Animal Husbandry&Veterinary Medicine,2009,36(4):103-107.(in Chinese)
[16]唐建兵,李勤,陈璧,等.两种人毛囊外根鞘细胞培养方法的比较[J].广东医学,2005,26(1):44-46.TANG J B,LI Q,CHEN B,et al.A comparison of two different methods for the culture of human hari follicle outer root sheath cells[J].Guangdong Medical Journal,2005,26(1):44-46.(in Chinese)
[17]崔正军,陈壁,汤朝武,等.DispaseⅡ和胰酶消化法分离大鼠皮肤角朊细胞的对比研究[J].第四军医大学学报,1998,19(3):349-350.CUI Z J,CHEN B,TANG C W,et al.Comparative study of rat skin keratinocytes isolated DispaseⅡand trypsin digestion[J].Journal of the Fourth Military Medical University,1998,19(3):349-350.(in Chinese)
[18]AASEN T,BELMONTE J C I.Isolation and cultivation of human keratinocytes from skin or plucked hair for the generation of induced pluripotent stem cells[J].Nat Protoc,2010,5(2):371-382.
[19]CUI Z F,HU Y X,WANG H,et al.Establishment and characterization of outer root sheath(ORS)cell line from Jining grey goat[J].Biotechnol Lett,2012,34(3):433-440.
[20]VIDAL V P I,CHABOISSIER M C,LUTZKENDORF S,et al.Sox9is essential for outer root sheath differentiation and the formation of the hair stem cell compartment[J].Curr Biol,2005,15(15):1340-1351.
[21]OHYAMA M,TERUNUMA A,TOCK C L,et al.Characterization and isolation of stem cell-enriched human hair follicle bulge cells[J].J Clin Invest,2006,116(1):249-260.
[22]HSU C K,YANG C C,SHIEH S J.Skin stem cells and their roles in skin regeneration and disorders[M]//WARBURTON D.Stem cells,Tissue engineering and regenerative medicine.New Jersey:World Scientific Publishin,2014:125.
[23]李玉荣,范文斌,李长青,等.内蒙古绒山羊次级毛囊组织形态周期性变化研究[J].中国农业科学,2008,41(11):3920-3926.LI Y R,FAN W B,LI C Q,et al.Histomotphology research of the secondary follicle cycling of Inner Mongolia cashmere goat[J].Scientia Agricultura Sinica,2008,41(11):3920-3926.(in Chinese)
[24]FUJIE T,KATOH S,OURA H,et al.The chemotactic effect of a dermal papilla cell-derived factor on outer root sheath cells[J].J Dermatol Sci,2001,25(3):206-212.
[25]LI W,LU Z F,MAN X Y,et al.VEGF upregulates VEGF receptor-2on human outer root sheath cells and stimulates proliferation through ERK pathway[J].Mol Biol Rep,2012,39(9):8687-8694.
[26]COMPOSTO G,KIM S,HECK D,et al.Nitrogen mustard induces DNA damage and structural changes in mouse skin hair follicles[J].FASEB J,2015,29(S1):766.7.
[27]OTA Y,SAITOH Y,SUZUKI S,et al.Fibroblast growth factor 5inhibits hair growth by blocking dermal papilla cell activation[J].Biochem Biophys Res Commun,2002,290(1):169-176.
[28]SUZUKI S,KATO T,TAKIMOTO H,et al.Localization of rat FGF-5protein in skin macrophage-like cells and FGF-5Sprotein in hair follicle:Possible involvement of two Fgf-5gene products in hair growth cycle regulation[J].J Investig Dermatol,1998,111(6):963-972.
[29]DROGEMULLER C,RUFENACHT S,WICHERT B,et al.Mutations within the FGF5gene are associated with hair length in cats[J].Anim Genet,2007,38(3):218-221.
[30]HOUSLEY D J,VENTA P J.The long and the short of it:Evidence that FGF5is a major determinant of canine‘hair’‐itability[J].Anim Genet,2006,37(4):309-315.
[31]HEBERT J M,ROSENQUIST T,GOTZ J,et al.FGF5as a regulator of the hair growth cycle:Evidence from targeted and spontaneous mutations[J].Cell,1994,78(6):1017-1025.
[2]MULLER-ROVER S,FOITZIK K,PAUS R,et al.A comprehensive guide for the accurate classification of murine hair follicles in distinct hair cycle stages[J].J Investig Dermatol,2001,117(1):3-15.
[3]KUMAMOTO T,SHALHEVET D,MATSUE H,et al.Hair follicles serve as local reservoirs of skin mast cell precursors[J].Blood,2003,102(5):1654-1660.
[4]KRAUSE K,FOITZIK K.Biology of the hair follicle:The basics[J].Semin Cutan Med Surg,2006,25(1):2-10.
[5]PENA J C,KELEKAR A,FUCHS E V,et al.Manipulation of outer root sheath cell survival perturbs the hair‐growth cycle[J].EMBO J,1999,18(13):3596-3603.
[6]PAUS R,COTSARELIS G.The biology of hair follicles[J].New Engl J Med,1999,341(7):491-497.
[7]LENOIR M C,BERNARD B A,PAUTRAT G,et al.Outer root sheath cells of human hair follicle are able to regenerate a fully differentiated epidermis in vitro[J].Dev Biol,1988,130(2):610-620.
[8]李京佳,林相国,许涛,等.VEGF家族及其在肿瘤生长中作用的研究[J].现代生物医学进展,2012,12(4):777-779,701.LI J J,LIN X G,XU T,et al.VEGF and the role of VEGF in tumor growth[J].Progress in Modern Biomedicine,2012,12(4):777-779,701.(in Chinese)
[9]蔡源源.血管内皮生长因子的调控及其作用研究进展[J].组织工程与重建外科杂志,2011,7(1):51-54.CAI Y Y.Advances in the regulation and role of VEGF[J].Journal of Tissue Engineering and Reconstructive Surgery,2011,7(1):51-54.(in Chinese)
[10]YANO K,BROWN L F,DETMAR M.Control of hair growth and follicle size by VEGF-mediated angiogenesis[J].J Clin Investig,2001,107(4):409-417.
[11]WU X J,ZHU J W,JING J,et al.VEGF165 modulates proliferation,adhesion,migration and differentiation of cultured human outer root sheath cells from central hair follicle epithelium through VEGFR-2activation in vitro[J].J Dermatol Sci,2014,73(2):152-160.
[12]袁超.绒山羊次级毛囊周期性变化相关microRNA的鉴定及miR-125b在毛乳头细胞中的功能研究[D].杨凌:西北农林科技大学,2014.YUAN C.Identification of microRNAs in secondary hair follicle cycling and functional analysis of miR-125bin dermal papilla cells of cashmere goats[D].Yangling:Northwest A&F University,2014.(in Chinese)
[13]周英昊,高晔,刘林丽,等.山羊y+L碱性氨基酸转运系统转录水平表达的研究[J].畜牧兽医学报,2015,46(8):1308-1316.ZHOU Y H,GAO Y,LIU L L,et al.Study on transcriptional level of y+L cationic amino acid transporter system in goat[J].Acta Veterinaria et Zootechnica Sinica,2015,46(8):1308-1316.(in Chinese)
[14]王丽,彭丽琴,张文彬,等.内蒙古白绒山羊皮肤毛囊发生发育规律的研究[J].畜牧兽医学报,1996,27(6):524-530.WANG L,PENG L Q,ZHANG W B,et al.Initiation and development of skin follicles in the inner mongolian cashmere goat[J].Acta Veterinaria et Zootechnica Sinica,1996,27(6):524-530.(in Chinese)
[15]刘志红,任立明,郭英,等.毛囊的周期性变化和分子调控及其在绒山羊上的研究进展[J].中国畜牧兽医,2009,36(4):103-107.LIU Z H,REN L M,GUO Y,et al.The cyclical variation and regulation of hair follicles and the study on cashmere goat[J].China Animal Husbandry&Veterinary Medicine,2009,36(4):103-107.(in Chinese)
[16]唐建兵,李勤,陈璧,等.两种人毛囊外根鞘细胞培养方法的比较[J].广东医学,2005,26(1):44-46.TANG J B,LI Q,CHEN B,et al.A comparison of two different methods for the culture of human hari follicle outer root sheath cells[J].Guangdong Medical Journal,2005,26(1):44-46.(in Chinese)
[17]崔正军,陈壁,汤朝武,等.DispaseⅡ和胰酶消化法分离大鼠皮肤角朊细胞的对比研究[J].第四军医大学学报,1998,19(3):349-350.CUI Z J,CHEN B,TANG C W,et al.Comparative study of rat skin keratinocytes isolated DispaseⅡand trypsin digestion[J].Journal of the Fourth Military Medical University,1998,19(3):349-350.(in Chinese)
[18]AASEN T,BELMONTE J C I.Isolation and cultivation of human keratinocytes from skin or plucked hair for the generation of induced pluripotent stem cells[J].Nat Protoc,2010,5(2):371-382.
[19]CUI Z F,HU Y X,WANG H,et al.Establishment and characterization of outer root sheath(ORS)cell line from Jining grey goat[J].Biotechnol Lett,2012,34(3):433-440.
[20]VIDAL V P I,CHABOISSIER M C,LUTZKENDORF S,et al.Sox9is essential for outer root sheath differentiation and the formation of the hair stem cell compartment[J].Curr Biol,2005,15(15):1340-1351.
[21]OHYAMA M,TERUNUMA A,TOCK C L,et al.Characterization and isolation of stem cell-enriched human hair follicle bulge cells[J].J Clin Invest,2006,116(1):249-260.
[22]HSU C K,YANG C C,SHIEH S J.Skin stem cells and their roles in skin regeneration and disorders[M]//WARBURTON D.Stem cells,Tissue engineering and regenerative medicine.New Jersey:World Scientific Publishin,2014:125.
[23]李玉荣,范文斌,李长青,等.内蒙古绒山羊次级毛囊组织形态周期性变化研究[J].中国农业科学,2008,41(11):3920-3926.LI Y R,FAN W B,LI C Q,et al.Histomotphology research of the secondary follicle cycling of Inner Mongolia cashmere goat[J].Scientia Agricultura Sinica,2008,41(11):3920-3926.(in Chinese)
[24]FUJIE T,KATOH S,OURA H,et al.The chemotactic effect of a dermal papilla cell-derived factor on outer root sheath cells[J].J Dermatol Sci,2001,25(3):206-212.
[25]LI W,LU Z F,MAN X Y,et al.VEGF upregulates VEGF receptor-2on human outer root sheath cells and stimulates proliferation through ERK pathway[J].Mol Biol Rep,2012,39(9):8687-8694.
[26]COMPOSTO G,KIM S,HECK D,et al.Nitrogen mustard induces DNA damage and structural changes in mouse skin hair follicles[J].FASEB J,2015,29(S1):766.7.
[27]OTA Y,SAITOH Y,SUZUKI S,et al.Fibroblast growth factor 5inhibits hair growth by blocking dermal papilla cell activation[J].Biochem Biophys Res Commun,2002,290(1):169-176.
[28]SUZUKI S,KATO T,TAKIMOTO H,et al.Localization of rat FGF-5protein in skin macrophage-like cells and FGF-5Sprotein in hair follicle:Possible involvement of two Fgf-5gene products in hair growth cycle regulation[J].J Investig Dermatol,1998,111(6):963-972.
[29]DROGEMULLER C,RUFENACHT S,WICHERT B,et al.Mutations within the FGF5gene are associated with hair length in cats[J].Anim Genet,2007,38(3):218-221.
[30]HOUSLEY D J,VENTA P J.The long and the short of it:Evidence that FGF5is a major determinant of canine‘hair’‐itability[J].Anim Genet,2006,37(4):309-315.
[31]HEBERT J M,ROSENQUIST T,GOTZ J,et al.FGF5as a regulator of the hair growth cycle:Evidence from targeted and spontaneous mutations[J].Cell,1994,78(6):1017-1025.